Effects of short-term treatment with various light intensities and hydroponic solutions on nitrate concentration of lettuce

Abstract

A glasshouse experiment was conducted to investigate the effects of five days’ treatment before harvest with various light intensities and seven hydroponic solutions on nitrate reduction in lettuce. The results showed that SPAD of new and expanded leaves were elevated by most of the hydroponic solutions under shading compared with the full nutrient solution, while no effect was demonstrated under non-shading. Additionally, lettuces under non-shading presented slightly higher leaf SPAD than those under shading. Nitrate concentrations of lettuce new leaves, expanded leaves and petiole were lowered significantly by all hydroponic solutions under shading and non-shading conditions compared with the full nutrient solution. Under non-shading, the efficiencies of seven hydroponic solutions varied largely, but no efficiency differences were showed under shading. N-free solution, 0.75 mM potassium sulphate solution and 5×10⁻⁵ mM ammonium molybdate solution functioned more efficiently in reducing nitrate concentration both in expanded leaves and petiole of lettuce before harvest. In conclusion, proper light intensity is a key important factor that determines the efficiencies of nitrogen-free hydroponic solution treatments in lowering nitrate concentrations in leaf blades and petioles of lettuce before harvest.

Keywords:

• Lettuce
• light intensity
• nitrate reduction
• petiole
• prior to harvest

Introduction

Green leafy vegetables, such as lettuce, cabbage and spinach, are known to be easier to accumulate nitrate in vegetables. Furthermore, compared with the vegetables grown in the open, the vegetables grown under cover were proved to accumulate higher nitrate concentration in tissues at inner low light intensity (Van Eysinga 1984, Premuzic et al. 2002), especially when heavy irrigation and fertilization were prevalent in practice. It has been estimated that approximately 80% of nitrate intake by the human body is from dietary vegetable consumption (Eichholzer and Gutzwiller 1998). Excessive intake of nitrate will pose a potential hazard to human health, especially for infants. The toxic effects of nitrate are due to its endogenous conversion to nitrite, which is implicated in the occurrence of methaemoglobinaemia, gastric cancer and many other diseases (Wolff and Wasserman 1972). Nowadays, it is a worldwide issue as to how to reduce nitrate concentration in leafy vegetables, which is more urgent for Chinese people with a high vegetable intake (Liu and Yang 2009).

Light intensity is a principal environment factor that dominates nitrate assimilation in leafy vegetables. High light intensity could enhance photosynthetic intensity, and therefore increase the supply level of nitrate assimilation materials, including reductants, energy and carbon skeleton (Appenroth et al. 2000). Therefore, low light intensity did result in nitrate accumulation since absorption rate of nitrate had exceeded the rate of nitrate assimilation (Steingröver 1986, Bloom-Zandstra et al. 1988). More importantly, overdue nitrate supply under low light intensity will aggravate nitrate accumulation in leafy vegetables because a large quantity of nitrate absorbed was stored in vacuoles for osmoregulation (Bloom-Zandstra and Lampe 1985). Based on this, Demšar et al. (2004) developed a computerized aeroponics system which could supply variable levels of nitrate by light-dependently changing nutrient solutions by which low nitrate lettuce was cultivated without yield reduction.

Besides light intensity, nutrient solution regulation is another effective measure to control nitrate concentration in leafy vegetables. Previous investigations have identified that some regulation measures during cultivation, e.g. nitrogen deprivation (Andersen and Nielsen 1992), partial nitrogen replacement (Gunes et al. 1994, 1996), osmotic regulation ions addition (Liu and Shelp 1996, Dong and Li 2003), use of biogas slurry (Liu et al. 2009a, 2009b) and functional elements (Mo), could decrease the nitrate contents in leafy vegetables. In addition, Mozafar (1996) found that pre-harvest transfer of spinach to N-free media reduced the nitrate concentration and increased the vitamin C content of the leaves by a substantial amount in several days. Also, Dong and Li (2003) suggested that adding Cl⁻, SO₄ ²⁻ into N-free nutrient solution could decrease nitrate concentration of pak choi. However, no investigation had been conducted to examine the efficiencies of substituting full nutrient solutions by simplified hydroponic solutions composed of osmotic regulation ions and functional elements prior to harvest in lowering nitrate concentration of leafy vegetables. The effects of light intensity on the efficiencies of alternative solutions before harvest in lowering nitrate concentration are also unclear. We hypothesized that the prior to harvest treatment efficiencies of the alternative solutions in nitrate reduction in leafy vegetables were various and light-dependent. In order to examine the hypothesis and select an efficient hydroponic solution for use before harvest to lower nitrate concentration of leafy vegetables in soilless cultivation, a glasshouse experiment was carried out. The objectives of the study were to compare the efficiencies of various hydroponic solutions in lowering nitrate concentration before harvest, and also to test the effects of light intensity created by shading.

Materials and methods

Experimental design

There were two light intensity treatments by shading (using a silver sunshade screen, hanging 30 cm above ground in the experimental glasshouse) and non-shading in the study. Under each light intensity treatment, eight hydroponic solution sub-treatments were designed (Table I). All above hydroponic solutions were prepared with distilled water and analytical reagents. The full nutrient solution is composed of 0.75 K₂SO₄, 0.5 KH₂PO₄, 0.65 MgSO₄, 0.1 KCl, 5.0 Ca (NO₃)₂, 1.0×10⁻³ H₃BO₃, 1.0×10⁻³ MnSO₄,1.0×10⁻⁴ CuSO₄, 5.0×10⁻⁶ (NH₄)₆M_O7O₂₄, 1.0×10⁻³ ZnSO₄, 0.1 EDTA-Fe (mM).

Table I. Sub-treatment codes and the corresponding hydroponic constitution.

Sub-treatment codes	Composition of hydroponic solutions for sub-treatment
1	Full nutrient solution (10 mM nitrogen)
2	Nitrogen-free nutrient solution
3	Phosphorus and potassium solution (0.75 mM K2SO4, 0.5 mM KH2PO4 and 0.1 mM KCl)
4	KCI (0.1mM) solution
5	K2SO4 (0.75 mM) solution
6	Ammonium molybdate solution (5×10^-5 mM)
7	NaCl solution (10 mM)
8	Microelement solution (the same concentrations as the full nutrient solution)

Lettuce transplantation, harvest and determination

Lettuce (Lactuca sativa L.), an Italian cultivar, was transplanted and cultivated in a cultivation bed (2 m×0.5 m×0.1 m) supplied with full nutrient solution after 20 days’ cultivation in a seedling tray. After 60 days’ cultivation, uniform lettuce plants were selected and transplanted into an experimental cultivation (length×width×height of each hydroponic pot is 32 cm×24 cm×11 cm) on 20 January 2010. Two lines and six holes were made on the lid with equal spacing, and a central one was used as a gas vent to aerate the nutrient solution with an air-pump (Atman EP-9000) to ensure the supply of soluble oxygen around the roots. Five lettuces in one hydroponic pot were planted. All sub-treatments were replicated three times. Experimental pots were randomly arranged. After five days’ growth, three plants for each replication were randomly harvested by removing the root. Lettuce shoot was separated into three parts, i.e. new leaf, expanded leaf and petiole for nitrate concentration determination. The SPAD of new and expanded leaves were determined with Minolta SPAD-502 chlorophyll meter (made in Japan). The nitrate concentration of fresh samples was determined as the method described by Liu et al. (2009a). The experiment was carried out in a glasshouse, with a temperature ranging from 15 to 30 °C. During the study period, all the days had clear weather. Calculated from the measured data (determined by AvaSpec-2048 fibre spectrometer, made in the Netherlands), the shading treatment decreased an average 54%±0.046% (n = 20) photosynthetically active radiation (µmol·m⁻²·s⁻¹) against the light intensity under the non-shading condition.

Statistical analyses

Variance analysis of data was conducted using SAS software 6.12. Significant differences between treatments were established by using the least significance difference (LSD) test.

Results and discussion

Leaf SPAD of lettuce treated with different hydroponic solutions and light intensities

SPAD value of leaves is used to indicate chlorophyll content and nitrogen nutrition level of vegetables. Compared with the full nutrient solution treatment, expanded leaf SPAD of lettuce was elevated for seven hydroponic solution treatments slightly or significantly under the shading condition (Table II). Among the hydroponic solutions, the lettuces treated with 5×10⁻⁵ mM ammonium molybdate solution had highest SPAD for both new and expanded leaf. However, hydroponic solution composed of 0.75 mM K₂SO₄, 0.5 mM KH₂PO₄ and 0.1 mM KCl decreased new leaf SPAD under the shading condition. The results also showed that no effect was demonstrated by the seven hydroponic solutions on the leaf SPAD in either expanded leaves or new leaves under non-shading condition. Leaf SPAD had been used with various crops as an indirect indicator of plant nitrogen status (Denuit et al. 1992, Gianquinto et al. 2003). Leaf SPAD is related to leaf chlorophyll content and nitrogen level. It may be attributed to the effects of no nitrogen supply and low light intensity on the chlorophyll turnover and leaf nitrogen nutrition. A previous study has shown that light intensity reduction increased leafy nitrogen content (Cantliffe 1973). In addition, nitrate content of rocket stored post-harvest at 5 °C under light (150 µmolm⁻²·s⁻¹ light with 12 h photoperiod) lowered, but the chlorophyll content did not show any significant reduction (Ferrante et al. 2003). Physiological mechanisms involved are still unclear and need further investigation.

Table II. Leaf SPAD of lettuce treated with different hydroponic solutions and light intensities.

	Shading		Non-shading
Treatments	Expanded leaves	New leaves	Expanded leaves	New leaves
1	14.6c	14.1b	19.6a	16.7a
2	16.4abc	14.1b	17.2a	16.5a
3	15.4bc	12.6c	18.7a	17.9a
4	16.9abc	15.3ab	17.2a	17.7a
5	17.3ab	15.6ab	17.9a	14.6a
6	18.6a	15.8a	19.0a	17.5a
7	16.4abc	14.2ab	17.5a	14.7a
8	17.2abc	14.7ab	18.6a	18.1a
In the same column, the different letters following the averages represent significant difference at p < 0.05.

Nitrate concentration of the new leaves, expanded leaves and petiole of lettuce treated with different hydroponic solutions and light intensities

Nitrate concentrations of lettuce new leaves were lowered significantly by 58.7% to 78.8% and 72.9% to 85.0% compared with the control under shading and non-shading conditions, respectively (Table III). Additionally, there was no efficiency variability among hydroponic solutions. Experimental data showed that nitrate concentrations of the expanded leaves and petiole of lettuce were impacted by the seven kinds of hydroponic solutions and light intensities. Under shading condition, nitrate concentration of expanded leaves and petiole of lettuce were decreased significantly, but there were no differences in nitrate concentration of both expanded leaves and petiole between seven hydroponic solution treatments. Under non-shading condition, nitrate concentration of expanded leaves and petiole of lettuce were dramatically lower than under shading condition. However, the decrease degree of nitrate concentration of both expanded leaves and petiole among seven hydroponic solutions varied largely. Additionally, efficiencies of seven hydroponic solutions in nitrate reduction of expanded leaves and petioles were relatively consistent. Among seven solutions, N-free solution, 0.75 mM potassium sulphate solution and 5×10⁻⁵ mM ammonium molybdate solution functioned more efficiently in reducing nitrate concentration in expanded leaves and petiole of lettuce.

Table III. Nitrate concentrations of the new leaves, expanded leaves and petiole of lettuce treated with different hydroponic solutions and light intensities (mg kg^-1).

	Shading			Non-shading
Sub-treatments	New leaves	Expanded leaves	Petioles	New leaves	Expanded leaves	Petioles
1	2658a	2902a	2509a	2197a	1684a	2350a
2	636b	1676b	1848b	466b	163c	666c
3	564b	1566b	1767b	334b	396bc	1085bc
4	749b	1390b	1751b	392b	563bc	1131bc
5	1097b	1232b	1692b	329b	270c	784c
6	915b	1468b	1869b	595b	306c	730c
7	889b	1576b	1980b	617b	521bc	926bc
8	1163b	1555b	1505b	374b	872b	1457b
In the same column, the different letters following the averages represent significant difference at p < 0.05.

Stopping nitrogen supply before harvest was an effective method to lower nitrate concentration in lettuce (Mozafar 1996). In the present experiment, seven simplified hydroponic solutions decreased nitrate concentrations in lettuce new leaves, expanded leaves and petioles together irrespective of shading or not. Additionally, the nitrate concentration was lowered more significant in three parts of lettuces under the non-shading condition than those under shading. We had supposed that shortage of other nutrient elements in nitrogen-free nutrient solution might impact normal photosynthesis, and further the nitrate reduction. This suggested that high light intensity was better for nitrate reduction under no nitrogen supply, and the nitrate remove efficiency depended on light intensity but the missed nutrient elements. Under low light intensity a significant increase in nitrate concentration in spinach tissues was found (Cantliffe 1973). The results indicated that these hydroponic solutions could replace nitrogen-free nutrient solution in prior to harvest regulation. However, a study found that distilled water had slight reduction effect on nitrate concentration of lettuce, but the efficiency was much inferior to K₂SO₄ and ammonium molybdate solutions (Liu and Yang 2009). Apparently, there were significant differences in nitrate reduction efficiencies between hydroponic solutions under non-shading condition, which might be related to the chemical compound constitution and physiological function in hydroponic solutions. To sum up, there are several physiological mechanisms to reduce nitrate concentration in lettuce under certain light intensities. One is osmotic ions, e.g. CI⁻, SO₄ ²⁻, which can replace nitrate anion existing in vacuoles. Thereby, nitrate stored in vacuoles was substituted by CI⁻, SO₄ ²⁻, and subsequently entered into cytoplasm and reduced and assimilated. Another is enzymatic activity regulation, like Mo, which can increase the activity of nitrate reductase. In current study, photosynthesis of lettuces was surely influenced by shortage of nitrogen and other nutrient elements, which might be a reason for the significant differences in nitrate reduction efficiency between hydroponic solutions.

Effects of shading on leaf SPAD and nitrate concentration in leaves and petioles

Shading treatment had significant effect on nitrate concentration in leaves and petioles of lettuce (n = 24) (Table IV).

Table IV. Effects of shading on leaf SPAD and nitrate concentration.

Treatments	SPAD of expanded leaves	SPAD of new leaves	Nitrate concentration in expanded leaves (mg kg^−1)	Nitrate concentration in new leaves (mg kg^−1)	Nitrate concentration in petiole (mg kg^−1)
Shading	14.6ab	16.6ab	1084a	1671a	1865a
Non-shading	16.7a	18.2a	663b	442b	969b
In the same column, the different letters following the averages represent significant difference at p < 0.05.

Meanwhile, leaf SPAD of expanded leaves and new leaves were slightly influenced by shading treatment. Whether shading or not, nitrate concentration in petiole was higher than new leaf and expanded leaf. Nitrate concentration in new leaf were lower than expanded leaves under shading condition, while there was no difference between new leaves and expanded leaves of lettuce under non-shading condition. However, nitrate concentrations in new leaf, expanded leaf and petiole of lettuce grown under shading were higher than the corresponding tissues of lettuce cultivated without shading. We hypothesized that the efficiencies of the prior to harvest regulation with hydroponic solutions were light-dependent. Our data verified that light intensity affected the efficiency of before harvest treatment with hydroponic solutions in lowering nitrate concentration in lettuce tissues, and also altered the nitrate distribution in various parts of lettuce.

Compared with nutrient solution adjustment during cultivation, prior to harvest treatment with alternative simplified hydroponic solutions is an effective method to control nitrate concentration in tissues of leafy vegetables with the advantages of high feasibility and efficiency. In soilless cultivation, waste nutrient solution release is avoidable for incomplete utilization of nutrients even in a closed recirculation cultivation system. Alternative hydroponic solutions put forward in this study are more environmentally friendly and resource-saving than nitrogen-free nutrient solution by removing nitrogen, phosphorus and other useless elements. Light intensity, osmotic ion and reductase activity regulation were feasible methods to lower nitrate concentration in leafy vegetables under the premise of nitrogen-free in practice for soilless cultivation in greenhouse and plant factory. In order to function more efficiently, the concentration range and interactions between effective chemical compounds should be investigated in future.

In the study, all nitrogen-free hydroponic solutions were effective in lowering nitrate concentration in lettuce during prior to harvest treatment, and their functional efficiency depended on the light intensity. Nitrogen-free solution, potassium sulphate solution and ammonium molybdate solution were good choices as short-term treating solution for nitrate reduction of soilless cultivated lettuce prior to harvest. From the view of environmentally friendly and resource-saving, potassium sulphate solution and ammonium molybdate solution were better than nitrogen-free solution in practice.

Acknowledgements

The authors want to thank the financial support of the Basic Scientific Research Fund of National Nonprofit Institutes (2009, BSRF201004), Institute of Environment and Sustainable Development in Agriculture, CAAS.